PROJECT SUMMARY: BET proteins (Brd2, Brd3, and Brd4) are chromatin-binding cofactors that have emerged as potent epigenetic drug targets in various cancers and inflammatory diseases. By modulating the chromatin environment, therapeutic BET protein inhibitors can succeed in what was largely considered impossible ? impacting the function of disease-associated transcription factors. However, unsurprisingly, BET inhibition is not completely benign, with early clinical studies reporting hematopoietic toxicities. Indeed, BET proteins are important in regulating housekeeping genes as well as in mediating inducible gene programs associated with cell fate decisions during embryological development and hematopoiesis. Their critical role in biology is highlighted by their conservation across species and their lethality in nullizygous mice. However, a clear mechanistic understanding of how BET proteins function similarly or distinctly is lacking. A major challenge has been a lack of models systems. Furthermore, though BET inhibitors are useful reagents, they lack specificity for individual BET proteins. The aim of this proposal is to gain deeper mechanistic insights into the roles of individual BET family proteins in regulating gene expression during Gata1-induced erythroid differentiation. Based on prior studies in which we observed that BET proteins Brd2 and Brd3 play a shared role in mediating erythroid differentiation that is distinct from that of Brd4, we hypothesize that BET proteins have both unique and overlapping roles in gene regulation that are conveyed by their modular functional domains. To test this, we will capitalize on a novel, gain-of-function cellular system that models erythropoiesis and for which rich genomic data sets already exist. Our studies aim to identify structural and mechanistic determinants of individual BET protein functions and elucidate any interdependencies that may exist. Our preliminary data indicate substantial genome-wide co-localization of Brd2 and the architectural protein CTCF. We will test the intriguing hypothesis that Brd2 in cooperation with CTCF plays an unforeseen role in three-dimensional genomic organization. We propose to explore the mechanistic underpinnings of this interaction and examine the functional consequences of Brd2-CTCF cooperativity with regard to genome folding at specific genes. In concert, the proposed studies aim to define the functions of individual BET proteins as guide for the design of selective BET inhibitors. Based on the novel discovery of a Brd2-CTCF interaction, existing pharmacologic BET inhibitors are expected to cause global perturbations in genome architecture. Dissecting the mechanism of Brd2 function in the context of CTCF might instruct the development of inhibitory compounds that avoid or selectively exploit perturbations in genome folding in therapeutic settings.